Tuberculosis and leprosy are known to be major public health problems. They are currently approximately 60.times.10.sup.6 individuals suffering from tuberculosis in the world (with an annual mortality of 3.times.10.sup.6), and approximately 15.times.10.sup.6 individuals suffering from leprosy. In France, approximately 10.sup.4 new cases of tuberculosis appear every year. Vaccination with BCG (Bacillus Calmette-Guerin, an attenuated strain of M. bovis) is far from effective in all populations. This efficacy varies approximately from 80% in Western countries such as England to 0% in India (results of the latest vaccination trial in Chingleput). The appearance of strains of M. tuberculosis resistant to the usual antituberculosis agents and the existence of mycobacterioses due to other, increasingly common mycobacteria such as M. avium, especially in patients with immunosuppression (AIDS in the largest number of cases), add to the urgency of developing a rapid method of detecting and identifying mycobacteria.
The diagnosis of tuberculosis and other related mycobacterioses is difficult to carry out; in effect the microorganisms responsible for these diseases are often present in small amounts, and when the amount of them is detectable by the methods conventionally used, the disease is already progressing and the patients are contagious to those around them. As a result of the very long generation time of these bacteria (24 h for M. tuberculosis compared with 20 min for E. coli) , culturing these organisms is difficult. Thus, it requires 6 to 8 weeks to identify the microorganisms, and longer to obtain an antibiogram usable for appropriate treatment of the patients. The need for a detection test not requiring culturing of the microorganisms, and directly usable with the pathological samples even when the microorganisms are present therein at low concentrations, is hence essential.
Several techniques are currently used in clinical practice for identifying a mycobacterial infection.
In the first place, direct detection of the microorganisms in the microscope should be mentioned; this technique is rapid, but does not permit identification of the mycobacterial species observed, and lacks sensitivity in as much as a large number of microorganisms have to be present in the sample (&gt;10.sup.4 /ml) in order to permit reliable detection (BATES J., CHEST, 1979, 76, (suppl.), 757-763).
Cultures, when positive, have a specificity approaching 100%, and permit identification of the mycobacterial species isolated; nevertheless, as specified above, growth of mycobacteria in vitro can be achieved only in the space of 3 to 6 weeks, and when few mycobacteria are present at the site of infection, repeated culturing is necessary in order to ensure a positive result (BATES J., 1979 and BATES J. et al., Am. Rev. Respir. Dis., 1986, 134, 415-417).
Serological techniques can prove useful under some conditions, but their use is limited by their low sensitivity and/or their low specificity (DANIEL T. M. et al., Am. Rev. Respir. Dis., 1987, 135, 1137-1151).
The presence or absence of mycobacteria may also be determined by hybridisation with DNA or RNA using probes specific for DNA sequences (KIEHN T. E. et al., J. Clin. Microbiol., 1987, 25, 1551-1552; ROBERTS M. C. et al., J. Clin. Microbiol., 1987, 25, 1239-1243; DRAKE T. A. et al., J. Clin. Microbiol., 1987, 25, 1442-1445). However, these methods also require culturing of the microorganisms.
Some DNA sequences of various mycobacteria, and in particular some genes coding for mycobacterial antigens have been described. There may be mentioned, in particular, PCT International Application WO 88/00,974, whose inventor is YOUNG R. and the content of which is recapitulated in a paper published in Nature, 1985, 316, 450; these publications describe the genes coding for five immunodominant antigens of M. leprae and, in particular, the gene coding for the 65-kD antigen has been sequenced. There may also be mentioned PCT International Application WO 88/05,823, whose co-inventors are HUSSON R., YOUNG R. and SHINNICK T. and the content of which is recapitulated in the paper published in J. Bact., 1987, 169, 1080-1088 and which describes the genes of M. tuberculosis coding for protein antigens, and in particular for the 65-kD antigen. This International Application specifies, in particular, that the genes of M. tuberculosis coding for five immunologically active proteins were isolated by systematic screening of a recombinant DNA library expressed in a bacteriophage lambda gt11, with a collection of monoclonal antibodies directed towards the protein antigens of this bacterium. One of the antigens of M. tuberculosis, a 65-kD protein possesses determinants commonn to M. tuberculosis and M. leprae.
PCT International Application WO 88/06,591, a co-inventor of which is, in particular, T. SHINNICK, describes a recombinant protein of 540 amino acids (65-kD protein) and also the DNA sequence and the vectors for the expression of the said protein, as well as the uses of the said recombinant protein. This Application also describes peptides corresponding to sequences of this protein and their uses.
Genes coding for proteins of other mycobacteria (M. africanum, M. smegmatis, M. bovis BCG and M. avium) have also been isolated. There may be mentioned, in particular, THOLE et al. (Infect. Immunol., 1987, 55, 1466-1475), who have described a 64-kD protein of M. bovis BCG expressed in E. coli.
However, the amounts of mycobacterial DNA present in most biological samples are insufficient to give a positive signal; this technique has hence proved unsuitable for the identification of mycobacterial DNA extracted directly from biological samples.
A number of studies have also shown some degree of structural homology between the different mycobacteria. However, differences in the DNA sequence of M. tuberculosis and M. bovis have been described in the 3' region of the open reading frame of the 65-kD antigen (SHINNICK et al., 1987, THOLE et al., 1987), but a homologous region has not been observed in the DNA of M. leprae (MEHRA et al., Proc. Nat. Acad. Sci. U.S.A., 1986, 83, 7013-7017, also PCT 88/000,974).
There are also publications which describe vaccines against mycobacteria, produced by genetic engineering; there may be mentioned, in particular, PCT International Application WO 88/02,027, which describes recombinant poxviruses capable of expressing mycobacterial antigens and which enable a protective immunological response to mycobacteria to be obtained.
The various detection methods of the prior art do not permit, on the one hand the detection and rapid identification of an Actinomycetales infection directly from a biological sample, and on the other hand the specific identification of groups, species or strains, which may even be present in small amounts.
The additional references which follow also constitute the state of the art prior to the present invention.
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